Fibrinogen is a blood plasma protein which is concerned in clot formation. It is converted into fibrin monomer by the action of the plasma protease thrombin. Fibrin monomers cluster together to form a weak clot and are then cross-linked by the action of activated factor XIII (i.e. factor XIIIa) to a form a stronger clot. Fibrinogen is used in therapy in combination with thrombin in the so-called fibrin sealants to achieve haemostasis, to seal wounds and for the controlled adhesion of tissue. Fibrinogen concentrates are also used for replacement therapy treatment of patients with fibrinogen deficiency (afibrinogenaemia) which may be inherited or acquired.
For all clinical applications, it is important to have highly pure fibrinogen in order to minimise any undesirable side effects resulting from, for example, the presence of unwanted contaminating proteins. In particular, it is desirable for fibrinogen preparations for clinical use to be free of plasminogen and plasmin (Blomback B., Blomback M., “Purification of human and bovine fibrinogen”, Arkiv for Kemi 1956; 10:415-443, and Mosesson M. W., “The preparation of human fibrinogen free of plasminogen”, Biochim Biophys Acta 1962, 57:204-213). Plasminogen is the inactive precursor of plasmin, a fibrinolytic enzyme which digests fibrin clots. Therefore the presence of plasminogen in a fibrinogen preparation intended for use in vivo is undesirable because any plasmin generated from the plasminogen at the site of clot formation may then destabilise the clot.
Plasminogen tends to co-purify with fibrinogen and its removal can be difficult. Some clinical fibrinogen preparations therefore contain anti-fibrinolytic agents to inhibit any plasmin or plasminogen present (e.g. aprotinin, a bovine protein inhibitor of plasmin; or tranexamic acid, a synthetic plasmin inhibitor also associated with neurotoxic side-effects). One advantage of separating plasminogen from fibrinogen is that there is then no need to use such fibrinolytic inhibitors in the clinical fibrinogen preparation.
In addition, it is highly desirable that fibrinogen derived from human or animal sources is treated to inactivate any blood-borne viruses which may be present, for example hepatitis virus or HIV. Various methods of virus inactivation are known in the art, including pasteurisation, dry heat treatment and solvent-detergent treatment (Pathogen Inactivation of Labile Blood Products, Council of Europe Expert Committee and Blood Transfusion Study Group on Pathogen Inactivation in Labile Blood Products, Transfusion Medicine, 2001, 11, 149-175).
Dry heat treatment is known to be effective for the inactivation of both enveloped and some non-enveloped viruses, whilst solvent-detergent treatment is known to be effective for the inactivation of enveloped (i.e. lipid coated) viruses such a hepatitis B.
Various methods for the purification of fibrinogen are known in the art. However, prior art purification methods suffer from various disadvantages. For example, precipitation methods do not allow easy incorporation of a solvent-detergent (SD) virus inactivation step, as the removal of SD reagents is much more efficiently effected chromatographically. Chromatography methods may not separate fibrinogen from plasminogen in a single step, which may lead to the need for additional chromatography to adsorb plasminogen, or the need to add an anti-fibrinolytic agent to the final fibrinogen preparation to combat residual plasminogen. In addition, not all the prior art methods are suitable for the purification of fibrinogen from a wide range of fibrinogen-containing solutions (including plasma and recombinant fractions).
U.S. Pat. No. 5,169,936 has previously suggested that immobilised metal ion affinity chromatography (IMAC) might be used in the preparation of human fibrinogen. However, no examples of such a method are disclosed, nor is there any suggestion that IMAC might be used for the separation of fibrinogen from plasminogen.
It is also known that the dissolution of fibrinogen concentrates can be difficult, and often requires the use of elevated temperatures or prolonged stirring (see U.S. Pat. No. 5,260,420 and EP-A 0804933). Due to the instability of liquid solutions of fibrinogen over time, fibrinogen preparations for clinical use are marketed either in the form of a deep-frozen solution or as a lyophilisate (i.e. a freeze dried preparation). Before use, the commercial product must be either thawed or reconstituted from the lyophilisate. Both these measures require significant time and effort.
It would therefore be advantageous to provide alternative methods for the purification of fibrinogen, in particular a method which is applicable to any fibrinogen containing starting material and which allows incorporation of one or more virus inactivation steps. It would also be advantageous to provide a method for the separation of fibrinogen from plasminogen. Furthermore, it would be advantageous to provide a lyophilised, and preferably heat treated, fibrinogen concentrate which can be readily redissolved at room temperature.